A visual system for evaluating CRISPR editing efficiency in cotton

Abstract Background Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) technology has advanced rapidly; however, current protoplast-based methods for evaluating genome-editing efficiency rely on expensive instruments and are incompatible with certain experimental strategies, highlighting the urgent need for a simple, cost-effective alternative. Results In this study, we developed a visual system for assessing genome-editing efficiency in cotton using GhF3H , a key gene involved in anthocyanin biosynthesis whose knockout results in distinct changes in tissue coloration. The red-background cotton line RZM24 was obtained via hybridization and backcrossing. Expression vectors harboring Cas9, Cas12a, or CasΦ and their corresponding single-guide RNAs (sgRNAs) targeting GhF3H were constructed and introduced into hypocotyls via Agrobacterium -mediated transformation, followed by callus induction. Successfully edited calli exhibited white or white-and-red chimeric phenotypes, whereas unedited calli remained red. Cas9 showed the highest editing efficiency, followed by Cas12a, with CasΦ showing the lowest. Genotyping confirmed that white and chimeric calli were capable of regenerating plants carrying heritable mutations, and the three Cas systems induced distinct mutation types. Conclusions This system enables rapid visual screening of editing events within 45 days, facilitating the comparison of CRISPR toolkits, validation of novel nucleases, and optimization of sgRNA delivery strategies. This approach may be adaptable to other crops and offers a practical solution to advance plant genome engineering.